T lymphocytes bearing AlphaBeta receptors respond to complexes of peptide antigen and class I or class II MHC molecules. The way in which protein antigens are transformed into peptides suitable for such binding, the events involved in facilitating such peptide-MHC association, and the intracellular pathways followed by MHC molecules both before and after peptide association are critical to our understanding of T cell immunity. We previously proposed two distinct pathways for peptide acquisition by class I and class II MHC molecules. To examine the biochemical and cell biological basis for this distinction, and the allele-independent molecular events involved in peptide-MHC molecule association, we have examined normal and mutant cells lines varying in their capacity to generate effective peptide-MHC complexes for the rate of class I and class II molecule assembly and transport, in the presence and absence of known MEC ligand peptides, and under varying growth conditions. In cells defective in transport of peptides into the ER, class I heavy chain-Beta2m complexes are unstable and inefficiently reach the cell surface. These loosely associated dimers can be accumulated at the cell surface by high concentrations of free Beta2m, revealing an important role for a dynamic equilibrium state that retards class I denaturation/ internalization. Free heavy chains on the membrane exist in a transiently re-foldable state, and the combination of peptide and Beta2m can create native class I molecules from these free heavy chains. Class II molecules are efficiently assembled and transported in these same cells, indicating no absolute requirement for ER peptides or invariant chain in promoting initial class II dimer assembly and ER egress. However, during intracellular transport, class II dimers alter their stability and conformation in concert with peptide acquisition in (a) post-Golgi compartment(s). This peptide acquisition is inefficient, and many "empty" class II molecules reach the cell membrane. Additional antigen increases the conversion of unstable to stable dimers and enhances overall class II expression. These latter observations provide new approaches for identifying the specific intracellular site(s) of class II peptide binding and for investigating the regulation of intracellular class II transport.